Tutorial

Check and clean sequencing reads

Filter low-quality reads with fastp and count what is left with seqtk, before and after. All in your browser; nothing is installed or uploaded.

What you will learn

  • Filter low-quality reads with fastp
  • Count reads and bases with seqtk size
  • Read the fastp report: what passed and why the rest failed
  • See how a stricter threshold changes the result

The data

One simulated sequencing run of 400 reads of 150 bp from bacteriophage PhiX174: 340 high-quality reads (base qualities 30 to 40) and 60 low-quality reads (base qualities 2 to 12), mixed together. Because it is simulated, you know how many reads should survive.

Tools in this tutorial

fastp
Checks and cleans FASTQ reads: removes low-quality reads, trims adapters and, for paired reads, merges pairs that overlap.
seqtk
A small toolkit for FASTA and FASTQ files: counts reads and bases, reports base composition, extracts regions and reverse-complements sequences.
Download run1.fastq (125 KB)

Watch it first

The whole tutorial in under 2 minutes, recorded in the live app. The run is sped up.

Steps

  1. 01

    Load the reads

    Open the Workflow Builder at biochef.app. In File manager, upload run1.fastq and tick it for the Input node. New to the editor? The map reads tutorial shows each move with screenshots.

  2. 02

    Count the reads before cleaning

    Search size and add Size (SEQTK). Connect the Input to it and add an Output node after it. seqtk size reports two numbers: reads and bases.

  3. 03

    Add fastp

    Select the Input again, search fastp and add Single-End QC (FASTP). Connect the Input to its in1 dot. Then select fastp, add a second Size (SEQTK) after it and an Output after that.

    Workflow: one Input feeding Size (SEQTK) directly and Single-End QC (FASTP), which feeds a second Size (SEQTK); each Size has an Output.
  4. 04

    Know the rules fastp applies

    Click the fastp node. With everything off, fastp uses its defaults: a base is good from quality 15 (qualified_quality_phred), and a read is dropped when more than 40% of its bases are below that (unqualified_percent_limit), when it has more than 5 N (n_base_limit) or when it is shorter than 15 bases (length_required).

    Single-End QC (FASTP) settings: qualified_quality_phred, length_required, n_base_limit, unqualified_percent_limit and trimming options, all switched off.
  5. 05

    Run and compare

    Press ▶. Click the first Output (before fastp), then the second (after).

    Output after fastp: 340 reads and 51000 bases.
  6. 06

    Read the fastp report

    Hover the ⓘ on the fastp node to see its report: how many reads passed, why the others failed, and the share of Q30 bases before and after filtering.

    fastp log: 400 reads before filtering, 340 passed, 60 failed due to low quality; Q30 bases rise from 85% to 100%.

What you should see

before fastp:  400   60000
after fastp:   340   51000

fastp keeps the 340 good reads and removes the 60 low-quality ones, so 9,000 bases go. In its report, Q30 bases rise from 85% to 100%.

Use the finished workflow

Import the finished workflow, data included, and press ▶. Remember that an exported workflow contains the data loaded in its Input nodes.

Download the finished workflow (JSON)

Exercise: what if the bar is too high?

On the fastp node, switch on qualified_quality_phred and type 35. Run again. Only 35 reads survive (5,250 bases): with this bar, many bases of the good reads (qualities 30 to 34) count as bad, and 365 reads fail. Filtering too hard throws good data away.

Show the solution
Output after fastp with qualified_quality_phred set to 35: 35 reads and 5250 bases.
Download the solution workflow (JSON)

From a test to a full run

The browser is the place to build and test a workflow on small data like this. For full datasets, the same workflow file is meant to run next to the data with the BioChef agent, which is in development. See Workflows for how the two fit together.

More tutorials: all BioChef tutorials.

This tutorial is licensed under CC BY 4.0. If you use BioChef in your work, please cite Rosa et al., BMC Bioinformatics 2026. BioChef is listed in bio.tools as biotools:biochef.